CCL21 is one of the chemokines which belong to the small molecule chemoattractive cytokine family. Chemokines mediate their chemical effect on target cells through G-protein-coupled receptors, which are characterized structurally by 7 transmembrane spanning domains and involved in the attraction and activation of mononuclear and polymorphonuclear leukocytes (25). CCL21 participated in three pathways, cytokine-cytokine receptor interaction, chemokine signaling pathway and NF-κB signaling pathway, based on the searches in KEGG database, and participated in apoptosis and survival_Lymphotoxin-β receptor signaling pathway based on the searches in GENEGO database. Besides, CCL21 plays roles in the regulation of ERK pathway in human non-small cell lung cancer cells (26). CCL21 is downregulated in many cancers and associated with lymph node metastasis, poor prognosis (27), apoptosis (26), cell cycle (28) and tumor growth (29). SPARCL1 belongs to SPARC family that contains ten protein members (30). SPARCL1 is widely expressed in normal and cancer tissues, and it was initially identified as an anti-adhesive extracellular matrix protein with anti-proliferative effects mediated through cell-cell adhesion (31,32). In addition, SPARCL1, which is downregulated in several tumor types such as colorectal and gastric cancer, is associated with tumor diagnosis, progression and prognosis (30,33,34). Therefore, SPARCL1 is considered to be a TSG (35) and may have many further unexplored functions in cancer development.

However, the studies on CCL21 and SPARCL1 associated with drug resistance are rare. Only one study reports that SPARCL1 is an extracellular matrix remodeling gene and may contribute to drug resistance in pediatric osteosarcoma (36). The research on CCL21 and SPARCL1 in ovarian cancer is limited. It is reported that CCL21 potentiated the cytotoxicity to ovarian cancer cells (37) and SPARCL1 is inactivated in ovarian cancer (38). More recently, CCL21 and SPARCL1 are noteworthy in ovarian cancer because they are silenced in the vast majority of high-grade serous ovarian adenocarcinomas (12).

Conserved protein sequence motifs are short stretches of amino acid sequence patterns that potentially encode the function of proteins (39). Except in CfCCL21, IL8 domain (accession: PF00048) was a unique and highly conserved motif in human CCL21 and its homologous proteins (Table I) according to SSDB Motif Search, indicating that IL8 domain might contribute to the function of CCL21. IL8 domain originally came from IL8 protein which closely related to drug resistance in many solid tumors and cancer cells. The upregulated expression levels of IL6 and IL8 may contribute to multidrug resistance in human breast cancer cells (40). Similarly, IL8 is overexpressed in paclitaxel resistance SKOV3 cells, and therefore is considered to be associated with paclitaxel resistance (41). These studies suggested that IL8 domain might closely relate to drug resistance, indicating that CCL21 might associate with drug resistance.

Four motifs comprising FOLN domain (accession: PF09289), Kazal_1 domain (accession: PF00050), Kazal_2 domain (accession: PF07648) and SPARC_Ca_bdg region (accession: PF10591) were highly conserved in human SPARCL1 and its homologous proteins in other species (Table II), suggesting that these motifs might closely relate to the functions of SPARCL1. Besides, efhand domain (accession: PF00036) was also observed in most SPARCL1 proteins. It has been reported that serine protease inhibitor Kazal-type 1, which contains Traw_N, Kazal_1 and Kazal_2 domains, affects multiple aggressive properties in breast cancer such as survival, invasiveness, and chemoresistance (42). Similarly, serine proteinase inhibitor Kazal-type 2, which contains only Kazal_1 and Kazal_2 domains, is also found to play an important role in tumor progression and response to the treatment in leukemia cell lines (43). These studies indicated that Kazal_1 and Kazal_2 domains might be associated with drug resistance in cancers. A previous study revealed that protein phosphatase with efhand domain may correlate with stress protective responses, cell survival, growth, proliferation and drug resistance (44). S100P with efhand domain is detected in a spectrum of human tumor cell lines and tissues derived from prostate, pancreas, breast, lung and colon, in which it is connected with malignant phenotype, hormone independence and resistance to chemotherapy (45). These studies indicated that efhand domain might also associate with drug resistance in cancers. In addition, on the basis of DOMINE online analysis, FOLN domain interacted with Kazal_1 domain, SPARC_Ca_bdg region interacted with FOLN and Kazal_1 domains and efhand domain interacted with Kazal_1 domain, suggesting that all these conserved motifs of SPARCL1 were closely related and interacted with each other. Taken together, we concluded that FOLN, Kazal_1, Kazal_2, SPARC_Ca_bdg, and efhand conserved in SPARCL1 were associated with drug resistance in cancers.

The involvement of CCL21 and SPARCL1 in cancer drug resistance had not been reported on the basis of literature co-occurrence, whereas there were 10 genes co-occurring with CCL21 and SPARCL1 in ovarian cancer (Fig. 1). Among those 10 genes, p53, BRCA1 and BRCA2 are well-known TSGs, and downregulation of these 3 genes contributes to the enhancement of drug resistance in ovarian cancer (5–8). ERBB2 and BCL2 are oncogenes; ERBB2 takes part in drug resistance in ovarian cancer (9), while BCL2 is reported to participate in drug resistance in other cancers (46,47). Besides, AFP is a drug resistance-related gene which plays a role in the expression of P-glycoprotein (48); TSC1 is a putative TSG participating in the signaling pathway of the mammalian target of rapamycin (mTOR) associated with proliferation, survival and drug resistance in leukemia cells (49); PTPRC, an apoptosis-related gene near cis-regulatory elements (50), is regarded as underexpression in breast cancer (51), suggesting that this gene may relate to drug resistance.

The involvement of CCL21 and SPARCL1 in ovarian cancer has rarely been studied. We observed that CCL21 had co-occurrences with p53, BCL2, PTPRC and ERBB2; SPARCL1 had co-occurrence with p53, BCL2, PTPRC and TSC1 (Fig. 1), suggesting that they might interact directly or indirectly. Taken together, we found that 8 in 10 genes which had co-occurrences with CCL21 and SPARCL1 in ‘ovarian cancer’ were drug resistance-related genes in ovarian and other cancers, suggesting that CCL21 and SPARCL1 might also be involved in drug resistance.

The functions of CCL21 and SPARCL1 were predicted using GeneMANIA (as shown in Fig. 2). CCL21 was co-expressed, co-localized, physically interacted, shared protein domains and pathways with a number of proteins, especially with CCL19, CCR7 and CCR6, suggesting that they were functionally related. In comparison, SPARCL1 had considerably fewer interactions with other proteins.

Based on the annotated functions in accordance with the GeneMANIA network (Table III), CCL21, together with other proteins, played important roles in the regulation of leukocytes, neutrophil chemotaxis, G-protein coupled receptor activity and calcium ion. It has been proven that leukocytes have close relationship with drug resistance, both in vivo and in vitro. In a ‘blinded’ study of 21 patients receiving combination cisplatin/carboplatin treatments, there was a direct relationship between DNA damage in leukocytes and disease response, and in leukocytes in vivo, persistence and accumulation are prominent features of the cisplatin-DNA adduct profile (52). Neutrophil chemotaxis seemed to be associated with drug resistance in an indirect way. For instance, celastrol is identified as an inhibitor of neutrophil chemotaxis, and it induces synergistic apoptosis when combined with conventional microtubule-targeting drugs and manifested efficacy toward taxol-resistant cancer cells at the cellular level (53). Similarly, stress and drug-induced interleukin-8 (IL8) signaling has been shown to confer chemotherapeutic resistance in cancer cells, while IL8 is a proinflammatory CXC chemokine contributed to the promotion of neutrophil chemotaxis and degranulation (54). G protein-coupled receptors essentially regulate all cellular processes, including those that are fundamental to cancer pathology, such as differentiation, proliferation, migration, tissue invasion, survival and drug resistance (55). Calcium content increases in multidrug resistant (MDR) cells and the resistance could be reversed by the calcium channel blocker verapamil, suggesting that calcium ion may play a role in drug resistance (56,57).

There was no annotated function for SPARCL1 based on GeneMANIA, but we could deduce its function through its interactions with other proteins. As shown in Fig. 2, SPARCL1 was co-expressed with many proteins such as RAMP3 and VWF. RAMP3 is associated with receptor-mediated endocytosis which is involved in drug resistance. Hsp47/CBP2 is a favorable candidate for targeted delivery of anticancer drugs in human squamous cell carcinoma of the head and neck, and the uptake of the targeted conjugate is inhibited in the presence of an anti-Hsp47 antibody, suggesting the involvement of active receptor mediated endocytosis in cell entry of the conjugate (58). The VWF is related to cell-substrate adhesion which is also involved in drug resistance. It has been proven that cell-substrate adhesion contributes to drug resistance via apoptosis in acute myeloid leukaemia, small cell lung cancer cells, breast cancer and glioblastoma cells (59).

In addition, CCL21 and SPARCL1 were co-expressed and interacted with each other indirectly through interacting with other proteins. IGFBP7 was found to be the most important one for CCL21 and SPARCL1 interactions. IGFBP7 had very strong physical interactions with CCL21 and shared protein domains with SPARCL1, indicating that CCL21, IGFBP7 and SPARCL1 might be functionally related. IGFBP7 has been identified as one of these factors responsible for the establishment and/or maintenance of oncogene-induced senescence, and has been shown to be a TSG in a variety of solid cancers (60). Aberrant expression of IGFBP7 in adult leukemia is correlated with chemotherapy resistance and shorter survival. Addition of IGFBP7 to leukemic cell lines inhibits cell growth without induction of apoptosis or senescence, suggesting a role of IGFBP7 in contributing to drug resistance through reduced sensitivity to cytostatic drugs (61).

The relationship of CCL21, SPARCL1 and small molecules were analyzed using BiologicalNetworks (Fig. 3). SPARCL1 had co-citation with only one small molecule, SPARC, which is the peptides of SPARC protein (SPARC_113–130 and SPARC_54–73) (62). SPARCL1 exhibits 62% identity with the anti-adhesive extracellular matrix protein SPARC, over a region of 232 aa spanning more than four-fifths of the SPARC coding sequence (63). SPARCL1 shared four domains (FOLN, Kazal_1, Kazal_2 and SPARC_Ca_bdg) with SPARC based on the SSDB Motif Search results (Table II). Thus, SPARCL1 might have similar functions with SPARC. SPARC is a candidate TSG and a putative resistance-reversal gene and plays an important part in drug resistance in ovarian cancer (64,65). Therefore, we concluded that SPARCL1, which is considered as a TSG (35), might also contribute to drug resistance in ovarian cancer.

CCL21 had co-citations with 12 small molecules, and half of them comprising omeprazole, SMS 201–995, adenosine monophosphate, ruthenium complex, hydrogen peroxide and 2,2′-bipyridine are associated with drug resistance in cancers. In vivo experiments show that oral pretreatment with omeprazole induces a sensitivity of human solid tumors to anticancer drugs (66). SMS 201–995 is proven to stimulate prostatic tumor growth and may sensitize tumor cells to subsequent chemotherapy (67). Adenosine monophosphate may participate in drug resistance of ovarian cancer through adenosine monophosphate-activated protein kinase pathway (68). The ruthenium complexes are effective tumor-inhibiting drugs in experimental therapy of autochthonous colorectal carcinomas in rats, and they can be promising candidate drugs in the second-line treatment of colorectal cancers resistant to other cytostatic drugs (69). Thioredoxin has much higher levels in all cisplatin-resistant human bladder and prostatic cancer cell lines compared with their drug-sensitive parental counterpart, and downregulation of its expression can increase cell sensitivity to cisplatin and also to other superoxide-generating agents including hydrogen peroxide, suggesting that hydrogen peroxide may also relate to drug resistance (70); 2,2′-bipyridine is a well-characterized chelating agent known to have anti-proliferative activity that links to drug resistance (71).

Total of 37 and 31 miRNAs were predicted to be the transcriptional targets of CCL21 and SPARCL1 through miRWalk, respectively. The pathway enrichment analysis of those miRNAs was performed with DIANA-mirPath, and an overview of the parts of the pathway modulated by miRNAs was integrated. Among all the pathways modulated by miRNAs targeted CCL21 and SPARCL1, 11 of them are involved in drug resistance in ovarian and many other cancers (Table IV).

Van Jaarsveld et al(72) systematically reviewed the miRNAs related to drug resistance in ovarian cancer. Among the miRNAs, some were the transcriptional targets of CCL21 and SAPRCL1 (Table IV). For instance, hsa-miR-125b and hsa-miR-370 were the targets of CCL21. Hsa-miR-125b is downregulated in paclitaxel resistant A2780 cell lines, therefore suggesting a direct involvement in the development of chemoresistance (73). Hsa-miR-370 is upregulated in platinum resistant EOC (74), suggesting that hsa-miR-370 may contribute to drug resistance through downregulation its target genes. Similarly, hsa-miR-431, the transcriptional target of SPARCL1, is also upregulated in topotecan resistant ovarian cancer cells (75). The pathway enrichment analysis of miRNAs related to drug resistance in ovarian cancer has also been studied. For example, it has been reported that 11 miRNAs are differentially expressed in cisplatin resistant ovarian cancer cells, which potentially target many important pathways comprising MAPK, Wnt signaling, mTOR signaling, apoptosis and many other signaling pathways which are all related to drug resistance in cancers (76).

All of these 11 drug resistance-related pathways modulated by the miRNAs targeted CCL21 and SPARCL1 (Table IV) were proven to be involved in drug resistance in ovarian cancer. The Wnt signaling pathway participates in drug resistance through inducing apoptosis and inhibiting tumor growth (77,78). Cell adhesion molecule is overexpressed in ovarian cancer, especially in recurrent/chemotherapy-resistant epithelial ovarian cancer, suggesting that cell adhesion molecule and its pathway may play a role in drug resistance (79). p53 signaling pathway is a well-studied and contributes to the whole process of cancer developments and it is involved in drug resistance in ovarian cancer through regulating cell proliferation following DNA damage (10). Cell communication is important to tumor mechanisms and relevant to the acquisition of drug resistance in ovarian cancer (80). With the better understanding of the relationship between cell cycle and the impact of chemotherapeutic agents on the cell cycle, it becomes apparent that this physiology can create drug resistance, therefore reducing combination chemotherapeutic efficacy (81,82). Amplified PI3K and activated Akt have been observed in 12–68% of tumors, and are closely associated with upregulation of mTOR signaling (83), therefore, activation of the PI3K/Akt pathway and its downstream mTOR signaling appear to represent drug resistance and poor prognosis (11,83); apoptosis plays an important role in the maintenance of physiological homeostasis in response to stimuli. When the apoptosis machinery fails, abnormal cells can survive, resulting in unopposed tissue growth and eventually fatal disease such as cancer (84). Apoptosis has been demonstrated to be involved in drug resistance in many solid tumors including ovarian cancer (85,86). VEGF signaling pathway is a key pathway in normal ovarian physiology and ovarian cancer, and closely related to drug resistance (87). Autophagy is involved in nucleus accumbens-1 mediated resistance to cisplatin, which is known to have important roles in proliferation, growth of tumor cells and chemotherapy resistance. Thus, the regulation of autophagy is considered to be involved in drug resistance in ovarian cancer (88). ABC transporters participated in drug resistance through controlling the drug transportation (89,90).

Taken together, we found that all the 11 pathways modulated by the miRNAs targeted CCL21 and SPARCL1 contributing to drug resistance in ovarian cancer, suggesting that CCL21 and SPARCL1 may also be involved in drug resistance in ovarian cancer.